Vector Identification and Gene Delivery Approach in Pigs

描述（由申请人提供）： 本提案的目的是开发一种分子变力性的新疗法，具体地说，将钙调节蛋白SERCA 2a（肌浆网Ca 2 <$ATPase）病毒介导的心肌递送至表现出心力衰竭的大型动物，以试图恢复功能并改善存活率而不恶化能量参数。充血性心力衰竭（CHF）是一个巨大的临床问题，需要有效的治疗方法。尽管传统的治疗方法取得了进展，包括药物治疗、心肌血运重建、机械辅助装置和移植，但CHF仍然是全球死亡的主要原因。因此，旨在降低CHF的发病率和死亡率并改善数百万患者的生活质量的新疗法特别有吸引力。心脏基因治疗在早期动物研究中显示出希望，并适用于心力衰竭的治疗。已知细胞内钙处理的缺陷是人类和实验性CHF中的关键异常。缺乏钙2？在来自人类和动物模型的衰竭心脏的松弛过程中，肌浆网（SR）的摄取与SRCa 2 + ATP酶（SERCA 2a）的表达和活性的降低有关。我们在过去五年的初步工作表明，1）基因转移是将SERCA 2a基因导入体外和体内肌细胞的有效手段，2）增加SERCA 2a的表达可恢复CHF啮齿动物模型的收缩性并使细胞内钙循环正常化。我们现在正在将实验从啮齿动物扩展到猪模型。我们的目的是1）确定病毒载体在从猪心脏分离的心肌细胞中的转导效率; 2）确定AAV载体和E1-E4缺失的重组腺病毒在猪体内基因转移后的转导效率; 3）检测近端人脑利钠肽（hBNP）启动子（相对于转录起始位点为-408至+100），以评价由压力超负荷相对于缺血诱导的能力。

DESCRIPTION (provided by applicant): The goal of this proposal is to develop a novel therapy for molecular inotropy, specifically, viral-mediated myocardial delivery of the calcium regulatory protein SERCA 2a (sarcoplasmic reticulum Ca 2¿ATPase) to large animals exhibiting heart failure in an attempt to restore function and improve survival without worsening energetic parameters. Congestive heart failure (CHF) represents an enormous clinical problem demanding effective therapeutic approaches. Despite advances in traditional approaches to its treatment, including pharmacologic management, myocardial revascularization, mechanical assist devices, and transplantation, CHF remains a leading cause of death worldwide. Therefore, a novel therapy aimed at decreasing the morbidity and mortality of CHF and improving the quality of life for millions of patients is particularly attractive. Cardiac gene therapy has shown promise in early animal studies and lends itself to the treatment of heart failure. A defect in intracellular calcium handling is known to be a key abnormality in both human and experimental CHF. Deficient Ca 2¿uptake by the sarcoplasmic reticulum (SR) during relaxation in failing hearts from humans and animal models has been associated with a decrease in the expression and activity of SR Ca2+ATPase (SERCA2a). Our preliminary work over the last five years have shown that 1) Gene transfer is an effective means of introducing the SERCA2a gene into myocytes in vitro and in vivo and 2) that increasing the expression of SERCA2a restores contractility and normalizes intracellular calcium cycling in a rodent model of CHF. We are now extending our experiments from rodents to porcine models. We aim to 1) determine the efficiency of transduction of viral vectors in cardiomyocytes isolated from pig hearts; 2) determine the efficiency of transduction of the AAV vectors and El-E4 deleted recombinant adenovirus following gene transfer in vivo in pigs; 3) test the proximal human brain natriuretic (hBNP) promoter (-408 to +100 relative to transcription start site) for the ability to be induced by pressure-overload versus ischemia.